CN105543269A

CN105543269A - Method for improving verticillium wilt resistance of plants by using Beauveria bassiana BbP4-ATPase gene

Info

Publication number: CN105543269A
Application number: CN201510738033.0A
Authority: CN
Inventors: 裴炎; 李玉杰; 陈杨; 范艳华; 侯磊; 李先碧; 宋水清
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2015-11-03
Filing date: 2015-11-03
Publication date: 2016-05-04
Anticipated expiration: 2035-11-03
Also published as: CN105543269B; WO2017076305A1

Abstract

The invention relates to a use of Beauveria bassiana BbP4-ATPase in improvement of the verticillium wilt resistance of plants, and a method for improving the verticillium wilt resistance of the plants. The Beauveria bassiana BbP4-ATPase gene is expressed in target plants to improve the verticillium wilt resistance of the plants. Detoxification of pathogen toxins is used to improve the resistance of the plants, and the pathogen race resistance characteristic does not exist. The method can be widely applied in improvement of the resistance of different pathogen physiologic races without the race-specific resistance characteristic.

Description

Beauveria bassiana BbP4-ATPase gene is utilized to improve plant to the method for resistance to verticillium wilt

Technical field

The invention belongs to plant genetic engineering field.Specifically, the method utilizing genetic engineering technique to improve disease resistance of plant is related to.

Background technology

Verticillium has a strong impact on the yield and quality of crop, and the loss that the whole world causes because of verticillium every year reaches multi-million dollar (Peggetal., 2002).Reporting that potato reduces more than 50% because infecting verticillium annual production, the underproduction (Powelsonetal., 1993 of 10-15% can be caused under normal circumstances; Roweetal., 1987,2002); Romaine lettuce growing area, the loss that verticillium causes is very easy to reach 100% (Subbaraoetal., 1997); Levinet (2003) etc. study discovery, and the underproduction that Fructus oleae europaeae causes because of verticillium can reach more than 70%; Verticillium is also the Main Cotton Diseases that majority plants cotton country, such as, the countries such as Australia, Brazil, Bulgaria, China, Greece, Peru, Turkey, Uganda, the U.S. and Uzbekistan, the underproduction (the Boleketal. of more than 30% cotton can be caused, 2005), fall ill serious area or the time underproduction can reach 100%.Verticillium is a kind of Major Diseases that restriction Cotton in China is produced, and the onset area in each cotton region of current China has accounted for plants more than 50% of the cotton total area, annual loss gined cotton 7.5-10 ten thousand tons, direct economic loss 16-20 hundred million yuan (Xiao Songhua etc., 2006).Increase year by year in Cotton in China verticillium grave illness plot, harm increases the weight of year by year, has become the current key constraints realizing cotton yield, stable yields.Verticillium not only causes a large amount of underproduction of crop and product quality to reduce, and the toxin that its pathogenic bacterium produce also can the health of harm humans.

Verticillium mainly infects by large beautiful branch bacterium VerticilliumdahliaeKleb and black and white Verticillium Verticilliumalboatrum a kind of soil-borne vascular bundle disease caused.Pathogenic bacteria, once in invaded plants body, does not just have effectively preventing method.Verticillium wilt pathogen host range is wide, 200 various plants comprising all dicotyledonss can be infected, pathogenic bacteria to survive about 20 years (Klostermanetal. with the form of Microsclerotia in soil, 2009), 1 is obtained for except the disease-resistant gene Ve of Solanum verticillium wilt pathogen except calendar year 2001 Kawchuk etc. clones from tomato, almost be not cloned into the disease-resistant gene from other plant, the Mechanism Study of Ve disease-resistant gene is shown that this gene only has resistance (Fradinetal., 2009) to the part microspecies of verticillium wilt pathogen.Early stage research is thought, after plant infection verticillium, the jellies that its conduit is formed by vegetable cell by spore, mycelia and pathogenic bacteria etc. block and cause the downright bad (Agrios that wilts, 2005), but research subsequently shows, catheter blockage is not the major cause that verticillium is wilted, along with deepening continuously of research, people recognize that the toxin that verticillium wilt pathogen produces is the important factor (Fradinetal., 2006) causing plant wilt.Also have some scholars to think, fascicular blocking is also the inductive effect (Chen Xusheng etc., 1998) based on toxin.

Facts have proved, utilize disease-resistant variety to be the unique cost-effective approach preventing and treating verticillium harm.Although traditional breeding way can utilize the resistant gene breeding resistant variety of crop itself or relationship kind, there is available resistant rice cultivar few, the shortcomings such as the seed selection time is long, and cost is large; Use chemical agent to be not only difficult to effectively prevent and treat verticillium but also contaminate environment.Along with plant gene engineering technology development and to plant and pathogen interactional understand in depth make by external source resistant gene import plant become an effective way to improve disease resistance.Not affinity phenomenon between the kind that can be broken traditions by transgenic technology in breeding, is eliminated crossing barrier, has greatly widened source and the application (GroverandGowthama, 2003) of resistant gene.

Utilize the Growth and reproduction of external source disease-resistant gene Product inhibiton verticillium wilt pathogen in plant body can improve the resistance of plant to verticillium, on the other hand, based on the pathogenesis of Amber box policy, improving plant to the detoxification ability of Amber box policy, is also improve plant to one of effective ways of resistance to verticillium wilt.But absolutely large number investigator mainly utilizes a kind of front method to reach the object improving plant resistance to environment stress, there is not yet successfully utilize the expression product of foreign gene to detoxify toxin that pathogenic bacteria produces and improve report to resistance to verticillium wilt.

Summary of the invention

One object of the present invention is to provide beauveria bassiana BbP4-ATPase gene raising plant to the purposes in resistance to verticillium wilt, by beauveria bassiana BbP4-ATPase gene integration target approach plant is built transgenic plant, and described BbP4-ATPase gene is made to express in plant and improve the resistance of plant to verticillium.

Another object of the present invention is that providing a kind of improves the method for plant to resistance to verticillium wilt, and by improving the resistance of described plant to verticillium at target plant expression in vivo foreign gene, described foreign gene is beauveria bassiana BbP4-ATPase gene.

More specifically, method of the present invention, comprises the steps:

Beauveria bassiana BbP4-ATPase gene integration target approach plant is built transgenic plant, and described BbP4-ATPase gene is expressed in plant.

Preferably, described method, comprises the steps:

1) build containing the recombinant plant expression vector from beauveria bassiana BbP4-ATPase gene;

2) described recombinant plant expression vector is imported in target plant, make beauveria bassiana BbP4-ATPase gene constitutive expression in target plant;

3) transgenic plant with the resisting verticillium of raising are obtained.

Preferably, the nucleotide sequence of described beauveria bassiana BbP4-ATPase gene is as shown in SEQIDNO.15.

The target plant that method of the present invention goes for is preferably tomato, tobacco or cotton.

In the inventive method, step 1) in recombinant plant expression vector there is structure as shown in Figure 8.

Another object of the present invention is to provide a kind of preparation method with the transgenic plant of resistance to verticillium wilt, comprises the following steps:

I) obtain beauveria bassiana BbP4-ATPase gene, and it is operationally inserted in plant expression vector, build plant expression vector;

Ii) by step I) plant expression vector that obtains transforms host, obtains transformant;

Iii) step I i is used) the transformant conversion of plant that obtains, obtain transgenic plant.

The object of the present invention is achieved like this: a kind ofly improve the method for plant to the sick resistance of verticillium, import containing the recombinant plant expression vector from the P class ATP enzyme gene of beauveria bassiana respectively in target plant, realize the constitutive expression of these genes in plant, improve transgenic plant to the detoxification ability of Amber box policy, and then obtaining the transgenic plant of resisting verticillium, the resistance of described transgenic plant to verticillium is better than target plant.

Particularly, the present invention utilizes beauveria bassiana BbP4-ATPase to improve plant to the method for resistance to verticillium wilt, comprises following step:

1) obtain beauveria bassiana BbP4-ATPase gene: introduce BamHI and SpeI restriction enzyme site design primer, then with beauveria bassiana cDNA for template carries out pcr amplification, amplified production is the BbP4-ATPase gene order of adding restriction enzyme site;

2) plant expression vector of constitutive expression BbP4-ATPase gene is built: BbP4-ATPase gene order amplification obtained inserts plant expression vector pLGN-35S-Nos, build a new plant expression vector, called after pLGN-35S-BbP4-ATPase;

3) genetic transformation of plant: utilize agrobacterium tumefaciens-mediated transformation, by above-mentioned steps 2) the pLGN-35S-BbP4-ATPase plant expression vector that obtains is integrated into Plant Genome, realize the constitutive expression of BbP4-ATPase gene in transgenic plant, improve transgenic plant to the resistance of verticillium;

4) acquisition of the BbP4-ATPase transfer-gen plant of resisting verticillium: by above-mentioned steps 3) transgenic plant that obtain carry out breeding further, Molecular Identification and anti-disease enzyme, obtain the BbP4-ATPase transfer-gen plant improved resistance to verticillium wilt.

Further, the step of the pLGN-35S-BbP4-ATPase plant expression vector construction of constitutive expression comprises: after obtaining the cDNA of beauveria bassiana, design upstream primer: 5 '- cGGGATCCaTGGCTGGACGACCTCCTGG (BamHI)-3 ', downstream primer 5 '- gACTAGTcTATTGCGGACGCGAGCTGG (SpeI)-3 ' carries out pcr amplification, amplified production and pLGN-35S-Nos vector plasmid carry out BamHI and SpeI double digestion respectively, and respectively reclaim enzyme cut after large fragment, recovery fragment connects by recycling ligase enzyme, build a new plant expression vector, called after pLGN-35S-BbP4-ATPase, to realize the constitutive expression of BbP4-ATPase gene in transgenic plant.

Raising plant provided by the present invention is to the method for resistance to verticillium wilt, that the BbP4-ATPase gene from beauveria bassiana is proceeded in tobacco, tomato and cotton cells respectively, realize the constitutive expression of these genes in transfer-gen plant, utilize the toxin that the P4 class ATP enzyme of exogene encodes removing toxic substances verticillium wilt pathogen produces in plant materials, improve transgenic plant to the detoxification ability of Amber box policy, and then improve transgenic plant to the disease resistance of verticillium.

The present invention is in conjunction with the characteristic of beauveria bassiana BbP4-ATPase mutant to cyclosporin A (CsA) sensitivity, simultaneously, CsA is a kind of lipophilic hydrophobicity cyclic peptide be made up of 11 amino acid, so also combine the similarity of CsA and Amber box policy part-structure, and the characteristic of verticillium wilt pathogen pathogenesis, creatively propose to utilize beauveria bassiana BbP4-ATPase gene constitutive expression in transgenic plant, improve transgenic plant to the detoxification ability of Amber box policy, and then improve transgenic plant to the strategy of the resistance of verticillium.

The present invention mainly utilizes transgene product to improve disease resistance of plant to the wither detoxification ability of verticillium toxin of Huang, and the detoxification ability further utilizing transgene product to improve plant against fungal toxin in an identical manner is also protection scope of the present invention.The plant expression vector related in the present invention simultaneously, transformant etc. are also in protection scope of the present invention.

The BbP4-ATPase transgene tobacco that the present invention obtains, T ₀hinder root filling bacterium liquid method for seedling utilization and inoculate high virulence L2-1 bacterial strain 30d, disease index is 34.87, and comparatively wild type control reduces by 40.78 respectively.Inoculation 30d, also has 9 transformants not occur illness in 38 BbP4-ATPase transformants.BbP4-ATPase transgene cotton T ₁for seedling, utilize leaching root inoculation method inoculation V991 defoliation verticillium bacterial strain 20d, non-transgenic reference disease index reaches 82.24, BbP4-TAPase transgene cotton has the disease index of 2 strains lower than 10, is respectively 8.33 and 4.55.The transgenic Fructus Lycopersici esculenti T utilizing the present invention to obtain ₀utilize for seedling plants and hinder root and fill with bacterium liquid method and inoculate high virulence L2-1 bacterial strain, the disease index that the disease index of non-transgenic tomato reaches 78.33, BbP4-ATPase transgenic Fructus Lycopersici esculenti is 44.44, and comparatively wild type control reduces by 33.89.Result of study shows, the transgene cotton utilizing the inventive method to obtain, tobacco and tomato significantly can put forward the disease resistance of transgenic plant to verticillium.Illustrate, raising plant provided by the invention is to the method Be very effective of resistance to verticillium wilt.The method is not only applicable to cotton, tobacco and tomato, and all energy all can utilize the resistance of the method raising to verticillium by the plant that verticillium wilt pathogen infects.

Method provided by the invention mainly utilizes the resistance removing toxic substances of pathogen toxin being improved to plant, does not thus have the feature of pathogenic bacteria microspecies resistance.Therefore, the resistance improving various pathogenic bacteria physiological strain can be widely used in, there is not the feature of little species specificity resistance.

Accompanying drawing explanation

Fig. 1 is the screening of CsA responsive type beauveria bassiana mutant

WT: beauveria bassiana wild type strain Bb0062; Control: beauveria bassiana T-DNA radom insertion transformant; Mu129 and Mu134:CsA responsive type beauveria bassiana T-DNA radom insertion transformant; CZA: Cha Shi solid medium; CsA: cyclosporin A; Scale=1cm.

Fig. 2 is BbP4-ATPase homologous recombination vector figure

Bar: careless fourth phosphine weedicide (phosphinothricin) resistant gene; PtrpC: the fungi composition promotor deriving from Aspergillus nidulans; TtrpC: the terminator of Aspergillus nidulans tryptophan synthetase gene; BbP4-ATPaseLB:BbP4-ATPase gene 5 ' flanking sequence; BbP4-ATPaseRB:BbP4-ATPase gene 3 ' flanking sequence; KanR: kalamycin resistance gene.

Fig. 3 is BbP4-ATPase homologous recombination vector plasmid enzyme restriction the result

M:DNA molecular weight standard Marker15,1:NcoI/BamHI endonuclease bamhi.

Cut pK2-BbP4-ATPaseLB-bar-BbP4-ATPaseRB plasmid vector with NcoI/BamHI enzyme, the object fragment of acquisition is 2Kb.

Fig. 4 is BbP4-ATPase gene disruption mutant PCR the result

M:DNA molecular weight standard Marker15; 1:pK2-BbP4-ATPaseLB-bar-BbP4-ATPaseRB plasmid; 2:BbP4-ATPase gene disruption mutant △ BbP4-ATPase; 3: heterologous recombination transformant; 4: beauveria bassiana wild type strain Bb0062.

Fig. 5 is the phenotype of BbP4-ATPase gene disruption mutant

WT: beauveria bassiana wild type strain Bb0062; Mu129:CsA responsive type beauveria bassiana T-DNA radom insertion transformant; △ BbP4-ATPase:BbP4-ATPase gene disruption mutant; CZA: Cha Shi solid medium; CsA: cyclosporin A.

Fig. 6 is pLGN-35S-Nos plant expression vector construction schema

LF-LF: recombinase recognition site (LoxpRT), for full genome synthesis forms; 35S-MCS-Nos, from pBINAR carrier (GenBank:AB752377.1), is obtained by PCR; NPT II gene is from the aphA2 on bacterial transposon Tn5; Gus gene is reporter gene, from intestinal bacteria.

Fig. 7 is pLGN-35S-BbP4-ATPase vector plasmid digestion verification result

M:DNA molecular weight standard Marker15; The BbP4-ATPase target fragment of the acquisition of BbP:pLGN-35S-BbP4-ATPase vector plasmid after enzyme is cut.

Fig. 8 is pLGN-35S-BbP4-ATPase plant expression vector figure

PLGN-35S-BbP4-ATPase plant expression vector figure, comprises the expression cassette of GUS::NPT II fusion gene that a 2 × 35S starts in this carrier, the expression cassette of the BbP4-ATPase gene of a 35S startup.

Fig. 9 is that BbP4-ATPase transgene tobacco GUS histochemical stain is analyzed

WT: the non-transgenic tobacco of wild-type; BbP:BbP4-ATPase transgene tobacco.

Figure 10 is the pcr amplification qualification partial results of BbP4-ATPase transgene tobacco GUS positive plant

Bp: base pair; M:DNA standard molecular weight DL2000; 1: Wild-type non-transgenic negative control; 2: plasmid positive control; 3-17:GUS stained positive transgenic tobacco plant.The pcr amplification result of BbP:BbP4-ATPase transgene tobacco GUS positive plant.

Figure 11 is inoculation verticillium wilt pathogen 30d, the phenotype of BbP4-ATPase transgenic tobacco plant

WT: the non-transgenic tobacco of wild-type; BbP:BbP4-ATPase transgene tobacco.

Figure 12 is that the expression level of foreign gene in BbP4-ATPase and VdP4-ATPase transgenic line and sick level compare

WT: wild type control; BbP-1, BbP-4 ... BbP-39:BbP4-ATPase transgene tobacco strain (independently transformant).

Figure 13 is that BbP4-ATPase transgene cotton GUS histochemical stain is analyzed

WT: the non-transgenic cotton of wild-type; BbP:BbP4-ATPase transgene cotton.

Figure 14 is the pcr amplification qualification partial results of BbP4-ATPase transgene cotton GUS positive plant

Bp: base pair; M:DNA standard molecular weight DL2000; 1: Wild-type non-transgenic negative control; 2: plasmid positive control; 3-17:GUS positive transgenic tobacco plant.BbP:BbP4-ATPase transgene cotton.

Figure 15 is for inoculation verticillium wilt pathogen 20d, BbP4-ATPase transgene cotton T1 are for the sick level of strain, sickness rate (%) and disease index

Null: the nontransgenic plants contrast be separated in transgenic line; B1, B10, B50, B53, B56, B57, B58, B6, B60 and B61:BbP4-ATPase transgene cotton independently transformant.

Figure 16 is inoculation verticillium wilt pathogen 20d, and BbP4-ATPase transgene cotton T1 is for the phenotype of seedling

WT: the non-transgenic cotton of wild-type; BbP:BbP4-ATPase transgene cotton; VdP:VdP4-ATPase transgene cotton.

Figure 17 is the expression level of BbP4-ATPase transgene cotton foreign gene

Figure 18 is that BbP4-ATPase transgenic Fructus Lycopersici esculenti GUS histochemical stain is analyzed

WT: wild type control; BbP:BbP4-ATPase transgenic Fructus Lycopersici esculenti.

Figure 19 is the pcr amplification qualification partial results of BbP4-ATPase transgenic Fructus Lycopersici esculenti GUS positive plant

Bp: base pair; M:DNA standard molecular weight; 1: Wild-type non-transgenic negative control; 2: plasmid positive control; The tomato plant of 3-17:GUS stained positive reaction.

Figure 20 is inoculation verticillium wilt pathogen 30d, the phenotype of plant

Embodiment

Below in conjunction with accompanying drawing, the present invention is described in further detail, but following explanation does not limit the present invention.

What the medicine and reagent in the invention process example was not specifically described is domestic conventional chemical reagent, equal reference " Molecular Cloning: A Laboratory guide " (Sambrook and Russell, 2001) that MATERIALS METHODS is not specifically described.

1, the proposition of invention technological core strategy

1.1BbP4-ATPase the acquisition of genetically deficient beauveria bassiana mutant

1.1.1CsA the acquisition of responsive type beauveria bassiana T-DNA insertion mutation body

The method of mutant library is built according to (2009) such as Luo, obtain beauveria bassiana T-DNA insertional mutagenesis library, and utilize the difference of mutant to CsA susceptibility to screen, with the CsA of 100 μ g/mL for screening pressure, obtain 2 CsA sensitive mutants, respectively called after Mu129 and Mu134 (Fig. 1).

Get 2 μ L beauveria bassiana conidial suspensions (1 × 10 ⁷individual/mL), point-like is inoculated in the CZA substratum of CZA (Cha Shi solid medium) and additional 100 μ g/mL cyclosporin A (CsA) respectively, carries out observing and taking pictures after 26 DEG C of cultivation 10d.

1.1.2 beauveria bassiana CsA tolerates the clone of genes involved

1) extraction of beauveria bassiana genomic dna

The extraction of beauveria bassiana genomic dna is carried out in (2002) filamentous fungus DNA extraction method slight changes such as reference side defends the country, and concrete operations are as follows:

1. inoculate beauveria bassiana (Beauveriabassiana) in 1/4SDY (Sharpe liquid nutrient medium) substratum, 26 DEG C, 200rpm, shaking culture 3 days, vacuum filtration collects mycelia;

2. liquid nitrogen flash freezer mycelia, grinding, every 100mg mycelia adds 300 μ LDNA extracting solutions (PH7.5Tris-HCl0.2M, NaCl0.5M, EDTA0.01M, SDS0.035M), and mixing, places 10min on ice;

3. add isopyknic chloroform: primary isoamyl alcohol (24:1), violent vortex 5min;

4.12000rpm, 4 DEG C, 10min is centrifugal, gets supernatant liquor, and the alkali phenol adding half volume carries out extracting, then adds chloroform isopyknic with alkali phenol: primary isoamyl alcohol (24:1) extracting;

5.12000rpm, 4 DEG C, 10min is centrifugal, gets supernatant liquor, adds isopyknic chloroform: primary isoamyl alcohol (24:1) extracting again.

6, repeating step 5, gets in supernatant liquor to new centrifuge tube;

7. add got supernatant liquor 2.5 times of volume ice bath dehydrated alcohols ,-80 DEG C of precipitation more than 30min;

8.10000rpm, 4 DEG C, centrifugal 10min collecting precipitation;

9.75% ethanol rinse precipitation, after vacuum-drying, uses ddH ₂o dissolution precipitation also processes with RNA enzyme, obtains beauveria bassiana genomic dna.

2) beauveria bassiana CsA tolerates the clone of genes involved

With beauveria bassiana genomic dna for template, utilize YADE method (Xiao Yuehua etc., 2002; Fangetal., 2005) T-DNA insertion point flanking sequence in Mu129 and Mu134 genome is cloned, find that destroyed in Mu129 and Mu134 is same gene by NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) comparison post analysis, i.e. phospholipid-translocatingP-ATPaseATPase, the albumen of this genes encoding has the similarity of about 60% with the P4-ATPaseDrs2 from yeast saccharomyces cerevisiae (Saccharomycescerevisiae), is therefore BbP4-ATPase by this unnamed gene.

1.1.3BbP4-ATPase the structure of gene disruption carrier

In order to the characteristic proving the CsA sensitivity that CsA sensitive mutant shows further is caused by BbP4-ATPase transgenation, the BbP4-ATPase gene disruption carrier building beauveria bassiana is verified.The displacement fragment of one section of sequence as homologous recombination is respectively got in the nucleotide sequence upstream and downstream of BbP4-ATPase gene.

Amplification BbP4-ATPaseLB fragment the primer is:

BbP4-ATPaseLB-F:5’- GGAATTCCGATGTCGTTCGAGTCGAAT-3’(EcoRI)(SEQIDNO.1)

BbP4-ATPaseLB-R:5’- GGAATTCGTGTAGGCGTTCAAGAATGA-3’(EcoRI),(SEQIDNO.2)

Amplification BbP4-ATPaseRB fragment the primer is:

BbP4-ATPaseRB-F:5’- GCTCTAGAAGCTTGCCATAATGTGCAAG-3’(XbaI)(SEQIDNO.3)

BbP4-ATPaseRB-R:5’- CCCAAGCTTTTCTTGGTGGTGTCGTAGGC-3’(HinDIII)(SEQIDNO.4)。

Amplification system is: 10 × LATaqPCRBuffer2.5 μ L, 25mMMgCl ₂2.5 μ L, 2.5mMdNTPMixture4 μ L, 100 μ g/ μ L beauveria bassiana genomic templates 2 μ L, 10 μMs of upstream and downstream primer each 1 μ L, LATaq5U/ μ L0.25 μ L, add water to 25 μ L systems.Amplification program is: 94 DEG C of 5min; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min30s, circulate 30 times; 72 DEG C of 10min.

Vector construction flow process is as follows:

1) obtain BbP4-ATPaseLB and BbP4-ATPaseRB fragment with beauveria bassiana genomic dna for template increases respectively, and reclaim pcr amplification product respectively;

2) by XbaI/HinDIII digestion step 1) glue that obtains reclaims BbP4-ATPaseRB product, and again reclaim enzyme cut after fragment, be built into the pK2-bar carrier utilizing same enzyme digest, acquisition pK2-bar-BbP4-ATPaseRB;

3) cut step 1 with EcoRI enzyme) glue reclaim BbP4-ATPaseLB product, again reclaim enzyme cut after fragment, this fragment is built into and utilizes same enzyme to carry out enzyme to cut and do in the pK2-bar-BbP4-ATPaseRB carrier of dephosphorylation process, obtain pK2-BbP4-ATPaseLB-bar-BbP4-ATPaseRB, by NcoI/BamHI digestion verification closure, cutting fragment, to be about 2kb be correct connection.The gene disruption Vector map built is shown in Fig. 2, and digestion verification the results are shown in Figure 3.

1.1.4BbP4-ATPase the acquisition of deletion mutant body and checking

Being utilized by the carrier pK2-BbP4-ATPaseLB-bar-BbP4-ATPaseRB built in 1.1.3 electric shocking method to import agrobacterium tumefaciens AGL-1, extract plasmid and carry out digestion verification, obtaining the AGL-1 transformant of result shown in Fig. 3 for transforming beauveria bassiana.With wild-type beauveria bassiana Bb0062 for acceptor, the method with reference to (2004) such as Fang carries out the genetic transformation of agriculture bacillus mediated beauveria bassiana, utilizes PCR to verify the transformant obtained, and checking the primer is MS-F:

5’-CGTAAGAGAATGTCGACCAT-3’/MS-R:

5 '-CTGAATCATCGACACGTCGT-3 ', homologous recombination transformant will amplify the fragment of 1862bp, and wild type strain amplifies the fragment of 789bp, and heterologous recombination transformant will amplify 1862bp/789bp two fragments.PCR amplification system is as follows: 10 × LATaqPCRBuffer2.5 μ L, 25mMMgCl ₂2.5 μ L, 2.5mMdNTPMixture4 μ L, 100 μ g/ μ L beauveria bassiana genomic templates 2 μ L, 10 μMs of upstream and downstream primer each 1 μ L, LATaq5U/ μ L0.25 μ L add water to 25 μ L systems.Amplification program is: 94 DEG C of 5min; 95 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min30s, circulate 30 times; 72 DEG C of 10min.Amplification as shown in Figure 4, is further analyzed checking to the phenotype of homologous recombination mutant simultaneously, and result display (Fig. 5), CsA sensitive mutants is consistent with BbP4-ATPase deletion mutant body surface type.Show, the CsA sensitive mutants that embodiment 1.1.1 obtains is BbP4-ATPase deletion mutant body.

Get 2 μ L beauveria bassiana conidial suspensions (1 × 10 ⁷individual/mL), point-like is inoculated in the CZA substratum of CZA and additional 10 μ g/mL cyclosporin A (CsA) respectively, carry out observing and taking pictures after 26 DEG C of cultivation 10d, BbP4-ATPase gene disruption mutant obtains consistent phenotype with Mu129 mutant.Scale=1cm.

The proposition of 1.2 invention technological core strategies

The result of study of embodiment 1.1.4 shows, beauveria bassiana BbP4-ATPase gene disruption mutant is comparatively responsive to CsA.BbP4-ATPase is a kind of membranin, participate in intracellular vesicle transport, so we infer, CsA may be transported to a certain privileged site in the mode of vesicle transport to carry out decomposing or utilizing (as vacuole), thus improve beauveria bassiana to the tolerance of CsA, and CsA is a kind of lipophilic hydrophobicity cyclic peptide be made up of 11 amino acid, its structure may with the similar of the pathogenic component of some of Amber box policy, therefore, in conjunction with verticillium wilt pathogen mechanism of causing a disease, the present invention creatively proposes the core strategy of this invention, " in transfer-gen plant, utilize the P class ATP enzyme of beauveria bassiana and verticillium wilt pathogen to the Amber box policy that detoxifies, and then improve transfer-gen plant to the resistance of verticillium ".

2, the clone of BbP4-ATPase gene

The extraction of 2.1 beauveria bassiana RNA and the synthesis of cDNA

Get appropriate beauveria bassiana wild type strain Bb0062 mycelia in 50mL1/4SDY (1/4 Sharpe liquid nutrient medium) substratum, 200rpm, 26 DEG C of shaking culture 3 days, suction filtration, collection mycelia, utilize liquid nitrogen grinding method, extract the total serum IgE of beauveria bassiana by the EASYspinRNA rapid extraction test kit working instructions of Aidlab company, the RNA of acquisition utilizes 1% agarose electrophoresis Detection job.Recycling TaKaRA company rTreagentKitwithgDNAEraser test kit synthesis cDNA, operates strict by specification and carries out.

The clone of 2.2BbP4-ATPase gene

The beauveria bassiana cDNA obtained with embodiment 2.1 for template, with BbP4-ATPase upstream region of gene primer 5 '- cGGGATCCaTGGCTGGACGACCTCCTGG (BamHI)-3 ' and downstream primer 5 '- gACTAGTcTATTGCGGACGCGAGCTGG (SpeI)-3 ' is primer, utilizes KOD-Plus-Neo (TOYOBO) enzyme to carry out pcr amplification, reclaims amplified production, obtains the BbP4-ATPase fragment with BamHI/SpeI restriction enzyme site.

PCR amplification system is: 10 × PCRBuffer5 μ L, 2mMdNTPs5 μ L, 25mMMgSO ₄3 μ L, 10 μMs of each 2 μ L of upstream and downstream primer, template (50 μ g/ μ L) 4 μ L, KOD-Plus-Neo1 μ L (1U/ μ L), add water to 50 μ L, amplification program is 94 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 4min30s, circulate 40 times.

3, the structure of pLGN-35S-BbP4-ATPase plant expression vector and the acquisition of Agrobacterium-mediated Transformation

The acquisition of 3.1pLGN-35S-Nos plant expression vector

PLGN-35S-Nos is the binary plant expression vector that this laboratory is transformed by pCambia2300.Its T-DNA section (between RB and LB region) replaces to the Reporter gene GUS of constitutive promoter CaMV35S-P control and the track fusion box of marker gene NPTII, and respectively with the addition of a LoxpFRT recombinase recognition site at these expression cassette two ends, and another expression cassette controlled by CaMV35S-P, detailed process is as shown in Figure 6.

The structure of 3.2pLGN-35S-BbP4-ATPase plant expression vector

The fragment BamHI obtain above-mentioned embodiment 2.2 and SpeI carries out double digestion, and reclaim endonuclease bamhi, carry out double digestion pLGN-35S-Nos plasmid with BamHI and SpeI simultaneously, and reclaim enzyme cut after large fragment, then T4DNA ligase enzyme is utilized to connect BbP4-ATPase fragment and the pLGN-35S-Nos fragment of recovery, connect product conversion bacillus coli DH 5 alpha, screening positive clone also carries out digestion verification (as shown in Figure 7), result shows, successfully BbP4-ATPase gene fragment is connected into pLGN-35S-Nos carrier, sequencing analysis is carried out according to carrier sequence design primer, the BbP4-ATPase gene order that sequence verification obtains is shown in SEQIDNO.15,-80 DEG C of preservation pLGN-35S-BbP4-ATPase plant expression vector positive colonies are for subsequent use, carrier has the structure shown in Fig. 8.

The acquisition of the recombinational agrobacterium of 3.3pLGN-35S-BbP4-ATPase plant expression vector

Utilize electrotransformation, pLGN-35S-BbP4-ATPase plant expression vector step 3.1 obtained proceeds to Agrobacterium LBA4404 competent cell, antibiotic-screening marker gene is utilized to carry out resistance screening, obtain positive colony, extract Agrobacterium plasmid again and carry out double digestion checking (the result and Fig. 7 digestion verification come to the same thing) with BamHI and SpeI, obtaining the recombinational agrobacterium containing pLGN-35S-BbP4-ATPase plant expression vector.

Extract intestinal bacteria or the Agrobacterium plasmid of pLGN-35S-BbP4-ATPase vector, BamHI and SpeI utilizes the sepharose of 1% to carry out electrophoresis after carrying out double digestion, obtain the object fragment of about 4080bp.

4, the Molecular Identification of transfer-gen plant

The GUS histochemical stain qualification of 4.1 transfer-gen plants

With reference to the method for (1987) such as Jefferson, with 9mm aperture, hole device is cut reconstituted tobacco, tomato or cotton plants young leaflet tablet and put GUS staining fluid (500mg/LX-Gluc, 0.1mol/LK ₃fe (CN) ₆, 0.1mol/LK ₄fe (CN) ₆, 1%TritonX-100 (v/v), 0.01mol/LNa ₂the 0.1mol/L phosphoric acid buffer of EDTA, pH7.0) in, 37 DEG C of insulation 2h.After dyeing, 75% ethanol decolorization, every 4h changes a destainer, until the color of non-coloured part is taken off completely.Blade does not all have the blue twice-laid stuff occurred to be nontransgenic plants, and what dye blueness is transfer-gen plant.

The pcr amplification qualification of 4.2BbP4-ATPase transfer-gen plant

The tobacco that the GUS histochemical stain of all regeneration is positive, tomato and cotton plants, get its seedling leaves and be about 0.1g, schedule of operation to specifications, the plant genes group DNA rapid extraction test kit of Aidlab company is utilized to extract DNA, detect the quality of DNA with the agarose gel electrophoresis of 1% after obtaining DNA, then with the DNA extracted for template, whether the fragment of amplification BbP4-ATPase gene, all incorporate BbP4-ATPas gene to detect regeneration plant.

BbP4-ATPase gene PCR amplimer is: upstream primer: 5 '-AACAGGCCGATAATGCGCTA-3 '; Downstream primer: 5 '-GTCTCGGGAAGTCCTTGCTT-3 '.

25 μ L amplification systems comprise: 10 × LAPCRBuffer, 2.5 μ L; 25mmol/LMgCL ₂, 2.5 μ L; 2.5mmol/LdNTPMixture, 2 μ L; Template DNA, 1 μ L (about 10ng); 5 μm of ol/L upstream primers, 1 μ L; 5 μm of ol/L downstream primers, 1 μ L; LATaq enzyme, 0.2 μ L; ddH ₂o, 14.8 μ L.

PCR thermal cycler parameters: 94 DEG C, 5min; 94 DEG C, 30s, 57 DEG C, 30s, 72 DEG C, 30s, 30 circulations of increasing; 72 DEG C, 10min.The last amplified production agarose gel electrophoresis of 1% detects.

5, the acquisition of anti-disease enzyme pathogenic bacterium and the sick level statistical standard of plant

The acquisition of pathogenic bacterium of 5.1 anti-disease enzyme

The defoliation verticillium wilt pathogen V991 (Verticilliumdahliae of PDA solid medium activation, Scientia Agricultura Sinica research institute Plant Protection Institute, letter Gui Liang researcher is so kind as to give) or High pathogenicity L2-1 bacterial strain (Agricultural University Of Hebei, professor Ma Zhiying is so kind as to give), the a little mycelium inoculation of picking enters in CZB (Cha Shi liquid nutrient medium), 180rpm, 26 DEG C of shaking culture 7d, inoculate into CZB substratum in the ratio of 10% (bacterium liquid/CZB substratum) again, 180rpm, 26 DEG C of shaking culture 7d, filter with four layers of sterile gauze and remove mycelia in bacteria-removing liquid and impurity, deionized water adjustment verticillium wilt pathogen spore concentration reaches 10 ⁷individual/ml, this spore suspension is the pathogenic bacterium inoculation liquid of anti-disease enzyme inoculation.

The sick level statistical standard of 5.2 transfer-gen plants

With reference to 5 grades of sick level statistical methods of Young Cotton seedling diseases level of Bhat and Subbarao (1999), the sick level of statistics cotton, tobacco and tomato plant.

0 grade: plant appearance is without illness;

1 grade: the aobvious illness of plant leaf less than 1/3;

2 grades: plant leaf 1/3-2/3 shows illness;

3 grades: the aobvious illness of plant leaf more than 2/3;

4 grades: plant leaf all shows illness.

5.3 sickness rate and disease index calculation formula

6, gene transcript expression detects

The extraction of 6.1RNA and the synthesis of cDNA

Utilize plant RNA rapid extraction test kit (Aidlab product), extract plant RNA, the strict by specification of all operations carries out, and the RNA of acquisition utilizes 1% agarose electrophoresis Detection job.Recycling rTreagentKitwithgDNAEraser test kit (TaKaRa product) synthesizes cDNA, then is template amplification target gene with cDNA, and in RNA, the removal of plant genome DNA and all strict by specification of the synthesis of cDNA carry out.

6.2 gene transcript expression detect

Real-timePCR method is utilized to detect genetically modified expression level in plant.For homogenization cDNA concentration, cotton is interior mark with GhHIS3 gene, and tobacco is then interior mark with 18S gene.

20 μ L reaction systems comprise: cDNA1 μ L, and each 1 μ L, the 2 × iQSYBRGreenSupermix10 μ L of upstream and downstream primer, supplies 20 μ L with the distilled water without RNA enzyme.

Amplification program is: 95 DEG C of 3min that increase in advance; 94 DEG C of 10s, 56 DEG C of 30s, 72 DEG C of 30s, coamplification 40 circulation.Increased the rear relative expression quantity utilizing GeneStudy software analysis gene.

7, the genetic transformation of tobacco and the acquisition of transfer-gen plant

7.1 tobacco genetic transformation substratum

MSB:MS inorganic salt (MurashigeandSkoog, 1962)+B5 organic (Gamborg etc., 1968).

Seed germination medium: MSB+30g/L sucrose, adds 2.0g/LGelrite and is cured, pH6.0.

Dual culture substratum: MSB+30g/L sucrose+0.5mg/LIAA (indolylacetic acid)+2.0mg/L6-BA (6-benzyl aminopurine), adds 2.0g/LGelrite and be cured, pH5.4.

Calli induction media: MSB+30g/L sucrose+0.5mg/LIAA+2.0mg/L6-BA+400mg/LCef (cephamycin)+100mg/LKm (kantlex), adds 2.0g/LGelrite and be cured, pH5.8.

Young shoot inducing culture: MSB+30g/L sucrose+2.0mg/L6-BA, 200mg/LCef+100mg/LKm, add 2.0g/LGelrite and be cured, pH5.8.

Root media: MSB+30g/L sucrose+200mg/LCef, adds 2.5g/LGelrite and be cured, pH5.8.

The acquisition of 7.2 tobacco genetic transformation explants

Get ripe tobacco seed subnumber grain, the alcohol sterilizing 1min of 75%, incline alcohol fast; Chlorine bleach liquor's sterilizing 15min of 1%, period not failure of oscillation swing, to reach adequately disinfected object; Incline dechlorination acid sodium solution, utilizes aseptic double-distilled water rinsing seed 7-8 time.120rpm under aseptic condition, shaken at room temperature is cultured to seed germination, then proceeds to seed germination medium, is cultured to 4-6 sheet true leaf under 16h illumination/8h light culture condition.Get full-grown true leaf, super clean bench is cut into the leaf dish of 3-5mm Jie side as the explant transformed with knife blade under aseptic condition.

The preparation of 7.3 During Agrobacterium liquid

-80 DEG C of single bacterium colonies of the streak culture acquisition of agrobacterium strains containing pLGN-35S-BbP4-ATPase carrier preserved, picking list bacterium colony again, inoculate (5g/L sucrose in the YEB substratum of additional 50mg/LKm and 125mg/LSm (Vetstrep), 1g/L bacto-yeast extract, 10g/L bacterium tryptone, 0.5g/LMgSO ₄7H ₂o, pH7.0), 28 DEG C, 200rmp shaking culture spends the night, and when reaching 0.8 to bacterium liquid OD600 value, gets bacterium liquid centrifugal, it is resuspended that thalline adds same volume MSB liquid nutrient medium, and re-suspension liquid is the During Agrobacterium liquid transformed.

The genetic transformation of 7.4 tobaccos and plant regeneration

The leaf dish explant that step 7.2 obtains contaminates 30min in During Agrobacterium liquid prepared by step 7.3, hypsokinesis remove bacterium liquid, through the tobacco explants of agroinfection, be seeded in Dual culture substratum, 26 DEG C of light culture 2d, then subculture enters calli induction media, 14d follow-up substitution bud inducement substratum, cultivate about 14d again, cutting regenerated green bud subculture enters in root media, is cultured to 2-3 leaf seedling in root media, cleans the agar around root, be transplanted in the nutrition pot of splendid attire turfy soil, and be positioned over greenhouse and cultivate.

The Molecular Identification of 7.5 transgene tobaccos and the acquisition of transfer-gen plant

Respectively GUS histochemical stain and pcr amplification qualification are carried out to the tobacco plant of regeneration according to the method for embodiment 6.It is all transfer-gen plants that all tobacco leaf GUS histochemical stain can obtain plant blue as shown in Figure 9.38 BbP4-ATPase transgene tobacco strains are obtained altogether through GUS histochemical stain qualification.In order to determine GUS histochemical stain identifies in the plant that is positive whether all incorporate BbP4-ATPase gene further, after the regeneration plant extraction blade STb gene of all GUS stained positive reactions, be template again with DNA, with synthesis sequence 9 and sequence 10 for primer, amplification BbP4-ATPase gene fragment, result shows, all BbP4-ATPase transgenosis reconstituted tobacco GUS positive reaction plant can be increased and be obtained the target fragment that BbP4-ATPase gene is about 240bp, (Figure 10), illustrate in GUS stained positive plant and all incorporate BbP4-ATPase gene.Through GUS histochemical stain and pcr amplification qualification, obtain independent transformation of 38 BbP4-ATPase transgene tobaccos altogether, all these transformants are all for anti-disease enzyme analysis.

With the DNA of transgene tobacco GUS stained positive reaction plant leaf for template, with SEQIDNO.9 and SEQIDNO.10 of the synthesis fragment that is primer amplification BbP4-ATPase gene, the plant of all GUS stained positive reactions can be increased the object fragment of the BbP4-ATPase gene obtaining about 240bp.

8, transgene tobacco is to the resistance of verticillium

When growing to 6-10 sheet leaf in the transgenic tobacco plant greenhouse that embodiment 7 genetic transformation and Molecular Identification obtain, knife blade is utilized to carry out hindering root process being about 1-2cm place from plant, then the L2-1 inoculation liquid 30mL (hinder root and fill with bacterium liquid method) of every strain pouring embodiment 5.1 preparation, puts the indoor cultivation of artificial climate on 22 DEG C of (night)-26 DEG C (daytimes).Inoculation 30d, by the sick level of the canonical statistics transfer-gen plant of embodiment 7.2.Result shows, and the average sick level of 38 independently BbP4-ATPase transformants is 1.39, and disease index is 34.87.And the average sick level of Wild-type non-transgenic contrast (WT lines of regeneration) is 3.03, disease index is 75.65 (tables 1), and compared with non-transgenic reference, the disease index of BbP4-ATPase transgenic line reduces 40.7.

Inoculation 30d, BbP4-ATPase transgene tobacco not only sick level and disease index is starkly lower than Wild-type non-transgenic contrast, and have 9 transformants in 38 BbP4-ATPase transformants, but not non-transgenic control lines is all seriously wilted, leaf chlorosis (Figure 11).Random selecting 5 transformants of not falling ill utilize axillalry bud to carry out expanding propagation, and then profit inoculates verticillium wilt pathogen in a like fashion, carry out anti-disease enzyme, result display inoculation 30 days, these transformants do not show obvious illness yet, confirm that the resistance of disease-resistant transgenic tobacco to verticillium obtained significantly improves further.Result shows, utilizes BbP4-ATPase gene can significantly improve the disease resistance of transgene tobacco to verticillium.

In order to the relation between the disease-resistant level raising of further clear and definite transgenic line and transgene expression level, random selecting 20 BbP4-ATPase transgene tobaccos are research object, the RNA of transgenic tobacco plant blade is extracted with the method for embodiment 6.1, and press the method synthesis cDNA of embodiment 6.1, be template amplification BbP4-ATPase gene fragment again with cDNA, then carrying out quantitative pcr amplification by the method for embodiment 6.2, is interior mark with 18SrRNA gene.Result shows, in all gene plants of walking around, BbP4-ATPase gene can carry out effective transcriptional expression, and the sick level of the higher transfer-gen plant of transcriptional expression level is lower, namely resistance and genetically modified transcriptional expression level also exist certain dependency, its genetically modified transcriptional expression level is higher, and transfer-gen plant is better to the resistance of verticillium.Expression level and the sick level of transgenic line are shown in Figure 12.

Concrete grammar: with transgene tobacco seedling leaves for material, extract the RNA of transfer-gen plant, and reverse transcription synthesis cDNA, take cDNA as template, with SEQIDNO.9 and SEQIDNO.10 of synthesis for primer amplification BbP4-ATPase gene, with the 18SrRNA gene of tobacco for internal standard gene, and increase for primer with SEQIDNO.11 and SEQIDNO.12 of synthesis, increased the rear relative expression quantity utilizing GeneStudy software analysis gene.

Verticillium wilt pathogen 30d inoculated by table 1, the sick level of BbP4-ATPase transgene tobacco and disease index

	Transformant sum (individual)	Do not fall ill and transform subnumber (individual)	Average sick level	Disease index
					WT	36	0	3.03	75.69
BbP4-ATPase	38	9	1.39	34.87

WT: WT lines; BbP4-ATPase:BbP4-ATPase transgenic tobacco plant.

38 BbP4-ATPase transgene tobacco transformants all utilize hinders root filling bacterium liquid method inoculation verticillium wilt pathogen spore suspension (10 ⁷individual spore/mL), inoculation 30d, adds up the sick level of each transformant, then calculates average sick level and the disease index of all transformants and contrast respectively by 5 grade standards of 0-4 level.Non-transgenic reference is all seriously fallen ill, and average sick level reaches 3.03, and disease index reaches 75.69, and the average sick level of BbP4-ATPase transgenic line is 1.39, and disease index is 34.87.

9, the genetic transformation of cotton:

9.1 Cotton Transformation substratum

Minimum medium: MSB (+B5 is organic for MS inorganic salt);

Seed germination medium: 1/2MSB+20g/L sucrose+6g/L agar, tap water is prepared, natural pH;

Dual culture substratum: MSB+0.5mg/LIAA+0.1mg/LKT (6-Furfurylaminopurine)+30g/L glucose+100 μm of ol/L Syringylethanone+2.0g/LGelrite, pH5.4;

The de-bacterium culture medium of screening: MSB+0.5mg/LIAA+0.1mg/LKT+75mg/LKm+500mg/Lcef+30g/L glucose+2.0g/LGelrite, pH5.8;

Calli induction media: MSB+0.5mg/LIAA+0.1mg/LKT+75mg/LKm+200mg/Lcef+30g/L glucose+2.0g/LGelrite, pH5.8;

Embryo callus subculture inducing culture: MSB+0.1mg/LKT+30g/L glucose+2.0g/LGelrite, pH5.8;

Fluid suspension culture base: MSB+1.91g/L saltpetre+0.1mg/LKT+30g/L glucose, pH5.8;

Body embryo maturation medium: MSB+15g/L sucrose+15g/L glucose+0.1mg/LKT+2.5g/LGelrite, pH6.0;

Seedling substratum: SH+0.4g/L activated carbon+20g/L sucrose, pH6.0.(Schenk&Hildebrandt，1972)

The acquisition of 9.2 transformation explant

Upland cotton seed shells, seed benevolence 0.1% mercuric chloride sterilizing 10min, after aseptic water rinsing 5-6 time, is inoculated in seed germination medium, 28 DEG C of light culture 5-7d.Aseptic hypocotyl is cut into the long segment of 3-5mm, as transformation explant.

9.3 transform the preparation with During Agrobacterium liquid

Transform with the preparation of Agrobacterium with embodiment 7.3, after the Agrobacterium bacterium liquid of cultivation is centrifugal, the Dual culture liquid nutrient medium of thalline interpolation same volume is resuspended, and re-suspension liquid is conversion During Agrobacterium liquid.

The genetic transformation of 9.4 Cotton Hypocotyls and the induction of embryo callus subculture

Explant During Agrobacterium immersion dye 20min, incline bacterium liquid, the bacterium liquid of explant excess surface is sucked again with aseptic filter paper, hypocotyl segment after dip-dye is inoculated in Dual culture substratum, 26 DEG C of light culture 2d, hypocotyl is seeded to the de-bacterium culture medium of screening, the calli induction media that the follow-up substitution of 20d adds kantlex (Km) and cephamycin (cef) carries out the induction of callus, interval 20d subculture once, 60d follow-up substitution embryo callus subculture inducing culture, fluid suspension culture is carried out after obtaining embryo callus subculture, to obtain the consistent embryo callus subculture of raised growth.

The induction of 9.5 body embryos and seedling are cultivated

The embryo callus subculture of fluid suspension culture, 30 order stainless steel sift net filtrations, the embryo callus subculture under sieve is inoculated into body embryo maturation medium dispersedly, and about 15d produces a large amount of body embryos, and its subculture is entered SH substratum, promotes the further seedling of body embryo.The regrowth of 3-4 leaf is transplanted into greenhouse and breeds.

9.6BbP4-ATPase the acquisition of transgene cotton and Molecular

With the cotton leaf of regeneration for material, respectively GUS histochemical stain and pcr amplification qualification are carried out to regeneration plant by the method for embodiment 4.The plant that GUS histochemical stain can obtain the blueness shown in Figure 13 is transfer-gen plant, be that a strain calculates to derive from the regeneration plant of an explant, identifying that the BbP4-ATPase transgene cotton of acquisition is the positive strains of 10 GUS through GUS histochemical stain, is 185 strain transfer-gen plants.For determining whether all incorporate BbP4-ATPase gene in GUS positive plant further, by the method for embodiment 4.2, pcr amplification qualification is carried out to transfer-gen plant, result shows, and the plant that all GUS histochemical stains are positive all can amplify the special band of target (Figure 14) of BbP4-ATPase (about 240bp) gene.In an identical manner to T ₁molecular Identification is carried out, through the transfer-gen plant T of Molecular Identification for seedling ₁for seedling all for disease-resistant analysis.

Concrete grammar: with the DNA of transgene cotton blade for template, with the fragment that SEQIDNO.9 and SEQIDNO.10 of synthesis is primer amplification BbP4-ATPase gene, the plant of all GUS stained positive reactions all can amplify the special band of target of BbP4-ATPase (about 240bp) gene.

10, transgene cotton is to the resistance of verticillium

Greenhouse breeding T ₀seed for plant results shells and stays seed benevolence, Aquaponic is carried out after 28 DEG C of sproutings, until there is true leaf in open and flat beginning of cotyledon, adopt the V991 defoliation verticillium wilt pathogen that leaching root inoculation method inoculation embodiment 5.1 obtains, root is transplanted in nutrition pot after infecting 2d, then 22 DEG C (night)-26 DEG C (daytime), 16h illumination, cultivate under 8h dark condition, after inoculation, interval 5d adds up the sick level of a plant by the sick grade standard of embodiment 5.2, inoculation 20d, the nontransgenic plants (Null) be separated in transgenic line is all fallen ill, disease index reaches 82.24, the disease index of 10 the transgenosis BbP4-ATPase strains obtained is all lower than non-transgenic reference, wherein BbP1, BbP10, BbP50, the sickness rate of 5 transgenic lines such as BbP53 and BbP58 is all lower than 50%, disease index is all lower than 30, wherein sickness rate is respectively 38.46%, 27.27%, 33.33%, 18.18% and 39.29%, disease index is respectively 23.08, 18.18, 8.33, 4.55 and 25.45 (Figure 15).The disease index of B50 and the B53 strain in these 5 strains all reaches disease-resistant level lower than 10, and all the other 3 strains reach resistance to sick level.Average sick level is utilized to compare, result shows, inoculation 20d, the average sick level of non-transgenic reference is 3.29, be susceptible to non-defoliation verticillium V991 strains expressed, the average sick level of these 5 strains of BbP1, BbP10, BbP50, BbP53 and BbP58 is respectively 0.92,0.73,0.33,0.18 and 1.02, to verticillium V991 strains expressed for anti-or resistance to.

Utilize leaching root inoculation method inoculation defoliation verticillium wilt pathogen V991 bacterial strain, inoculation 20d, adds up disease level by the sick grade standard of 0-5 level, and calculates sickness rate, disease index, and calculate the average sick level of each strain.Result shows, and the sickness rate of transgenic line, disease index and average sick level are all starkly lower than non-transgenic reference, show that the resistance of BbP4-ATPase transgene cotton to verticillium significantly improves.

Inoculation 20d, the typical illness of verticillium has all been there is in non-transgenic reference plant because verticillium wilt pathogen infects blade, the variable color of upper blade chlorosis, partial blade necrosis even come off, and the BbP4-ATPase transgene cotton true leaf leaf look that resistance improves is normal, plant strain growth is normal, just there is slight illness in indivedual plant cotyledon, has a little scab (Figure 16), significantly improve the resistance to verticillium.

In order to further illustrate the relation between transgene expression level and disease resistance, Real-timePCR is utilized to detect the transcriptional expression level of foreign gene.Take transgenic line as material, each strain gets three sample extraction RNA, and reverse transcription synthesis cDNA, then be template with cDNA, SEQIDNO.9 and SEQIDNO.10 is primer amplification BbP4-ATPase gene, with GhHIS3 gene for internal standard gene, amplimer is SEQIDNO.13 and SEQIDNO.14.Result display (Figure 17), in all BbP4-ATPase transfer-gen plants, BbP4-ATPase gene can effective transcriptional expression, compared with transgenic line disease index, the height of transgene expression level affects the resistance level of transgenic line, when transgene expression level is higher, the disease resistance of transgenic line is better.

11, the genetic transformation of tomato

11.1 tomato genetic transformation substratum

Minimum medium: MSB0 (organic+30g/L sucrose of the inorganic+B5 of MS, pH5.8).Solid medium adds agar (Murashige and Skoog, 1962 of 6g/L; Gamborg etc., 1968);

Dual culture substratum MSB1:MSB0+2.0mg/L6-BA+0.2mg/LIAA+100uMAS (Syringylethanone)+6g/L agar, pH5.4;

Screening culture medium MSB2:MSB1+500mg/Lcb (Pyocianil)+100mg/LKm+6g/L agar, pH5.8;

Subculture medium MSB3:MSB0+200mg/Lcb+100mg/LKm+6g/L agar, pH5.8;

Root media MSB4:MSB0+0.5mg/LIAA+200mg/LCef+50mg/LKm+6g/L agar, pH6.0.

The acquisition of 11.2 tomato genetic transformation explants

Micro-Tom tomato seeds 1% chlorine bleach liquor sterilizing 10-15min, then aseptic water rinses 5-6 time, be seeded on MSB0 substratum, 25 DEG C, photoperiod of 16h illumination/8h dark is in sprouting about 7d, the open and flat aseptic seedlings cotyledon of robust growth cuts two ends, and the part at middle part about 2/3 gives over to the explant of Agrobacterium tumefaciens-mediated Transformation.

11.3 transform the preparation with During Agrobacterium liquid

Transform with the preparation of Agrobacterium with embodiment 7.3, after the Agrobacterium bacterium liquid of cultivation is centrifugal, the MSB0 liquid nutrient medium of thalline interpolation same volume is resuspended, and re-suspension liquid is conversion During Agrobacterium liquid.

11.4 tomato genetic transformation

With reference to the method for (2004) such as Cortina, to grow the cotyledon of about 7d aseptic seedling for explant, agrobacterium tumefaciens-mediated transformation is utilized to carry out genetic transformation.

Bacterium liquid is removed in During Agrobacterium immersion dye explant 10min hypsokinesis, and aseptic thieving paper sucks explant excess surface bacterium liquid, then inoculates the Dual culture substratum MSB1 into being covered with one deck aseptic filter paper, 25 DEG C of dark Dual culture 2d.After Dual culture completes, inoculated by explant in screening culture medium MSB2 and carry out differentiation culture, 25 DEG C, photoperiod of 16h illumination/8h dark cultivates 2 weeks, then explant subculture is entered the generation of MSB3 substratum callus induction, every 2 weeks subcultures once.After producing Km resistance young shoot, young shoot is cut and inoculates into MSB4 root media, obtain Km resistance regeneration plant.The vegetative seedling of the long 3-5cm of root, is transplanted into greenhouse-grown seedling.

The Molecular Identification of 11.5 transgenic Fructus Lycopersici esculentis and the acquisition of transfer-gen plant

Respectively GUS histochemical stain and pcr amplification qualification are carried out to the tomato plant of regeneration according to the method for embodiment 6.It is all transfer-gen plants that all tomato leaf GUS histochemical stain can obtain plant blue as shown in figure 18, 27 strain BbP4-ATPase Transgenic Tomato Plants are obtained altogether through GUS histochemical stain qualification, in order to determine GUS histochemical stain identifies in the plant that is positive whether all incorporate BbP4-ATPase gene further, all regeneration plants extract blade STb gene, be template again with DNA, to synthesize SEQIDNO.9 and SEQIDNO.10 for primer, the fragment of amplification BbP4-ATPase gene, result shows, all GUS positive reaction plant can be increased and be obtained the special band of target (Figure 19) of BbP4-ATPase (about 240bp) gene.Pcr amplification result illustrates, all incorporates BbP4-ATPase gene in the BbP4-ATPase plant of GUS stained positive reaction.Through GUS histochemical stain and pcr amplification qualification, obtain 27 BbP4-ATPase transfer-gen plants altogether, all these transformants T ₀for plant all for anti-disease enzyme analysis.

12, transgenic Fructus Lycopersici esculenti is to the resistance of verticillium

Through the Transgenic Tomato Plants that embodiment 11 genetic transformation and Molecular Identification obtain, when growing to 4-6 sheet leaf in greenhouse, knife blade is utilized to carry out hindering root process being about 1-2cm place from plant, the root then L2-1 inoculation liquid 15mL for preparing of every strain pouring embodiment 5.1 is hindered in every strain symmetrically, puts the cultivation indoor cultivation on 22 DEG C of (night)-26 DEG C (daytimes).Inoculation 20d, by the sick level of the canonical statistics transfer-gen plant of embodiment 5.2.

Anti-disease enzyme result display (table 2), inoculation 30d, the average sick level of wild type control (WT lines of regeneration) is 3.13, and disease index is 78.33.The average sick level of 27 BbP4-ATPase transgenic Fructus Lycopersici esculenti transformants is 1.78, and disease index is 44.44, and compared with non-transgenic reference, the disease index of transgenic line reduces 33.89.Inoculation 30d, BbP4-ATPase transgenic Fructus Lycopersici esculenti not only sick level and disease index is starkly lower than non-transgenic reference, and have the sick level of 12 transformants in 27 BbP4-ATPase transformants not more than 1, the transfer-gen plant that resistance improves is that slight illness appears in the blade of bottommost, but not the sick level of non-transgenic control lines is all more than 3, and the blade of middle and upper part has started chlorosis flavescence (Figure 20).

Anti-disease enzyme result shows, utilizes BbP4-ATPase gene can significantly improve the disease resistance of transgenic Fructus Lycopersici esculenti to verticillium.

Verticillium wilt pathogen 30d inoculated by table 2, the sick level of transgenic Fructus Lycopersici esculenti and disease index

	Transformant sum (individual)	Sick level≤1 transforms subnumber (individual)	Average sick level	Disease index
					WT	15	0	3.13	78.33
BbP	27	12	1.78	44.44

CK: wild type control; BbP:BbP4-ATPase transgenic Fructus Lycopersici esculenti.

All plant utilize and hinder root filling bacterium liquid method inoculation verticillium wilt pathogen spore suspension (10 ⁷individual spore/mL), inoculation 30d, adds up the sick level of each transformant, then calculates average sick level and the disease index of all transformants and contrast respectively by 5 grade standards of 0-4 level.Non-transgenic reference is all fallen ill, and average sick level is 3.13, and disease index reaches the sick level of 78.33, BbP4-ATPase transgenic Fructus Lycopersici esculenti and disease index all significantly declines.

Reference

1. Chen Xu liter, Chen Yongxuan, Huang Junqi. secrete the biochemical characteristic of toxalbumin outside cotton verticillium wilt bacterial strain VD8. Jiangsu's agriculture journal, 1998,14 (2): 126-128.

2. side defends the country, Yang Xingyong, Zhang Yongjun, Pei Yan. a kind of rapid extracting method of fungal nucleic acid. and application and environmental organism journal, 2002,8 (3): 305-307.

3. Xiao Songhua, Wu Qiaojuan, Liu Jianguang, Di Jiachun, Xu Naiyin, Chen Xusheng. the creation of high-quality verticillium wilt-resistant cotton new germ plasm. Jiangxi cotton, 2006,6:10-14.

4. Xiao's moonlight, Luo Ming, side defends the country, and Roc is bright, Hou Lei, Luo little Ying, Pei Yan. and utilize YADE method to carry out cotton gene group PCR walking. Acta Genetica Sinica, 2002,29 (1): 62-66.

5.AgriosGN.,Plantpathology,5thedn..USA,UK:ElsevierAcademicPress,2005.

6.AnitaGroverandR.Gowthaman.,Strategiesfordevelopmentoffungus-resistanttransgenicplants.Science,2003,84:330-340.

7.Bhat，R.G.andSubbarao，K.V.,HostrangespecificityinVerticilliumdahliae.Phytopathology,1999,89：1218-1225.

8.FradinEF,BartPHJ,ThommaBPHJ.,PhysiologyandmolecularaspectsofVerticilliumwiltdiseasescausedbyV.dahliaeandV.albo-atrum.MolecularPlantPathology,2006,7:71–86.

9.GamborgO.L.,MillerR.A.,OjimaK.,Nutrientrequirementsofsuspensionculturesofsoybeanrootcell.ExperimentalCellResearch,1968,50:151-158.

10.JosephSambrookandDavidW.Russell.,MolecularCloning.ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,2001.

11.Kawchuk,L.M.,TomatoVediseaseresistancegenesencodecellsurface-likereceptors.ProceedingsoftheNationalAcademyofSciences,2001,98(11):6511-6515.

12.KlostermanSJ,AtallahZK,ValladGE,SubbaraoKV.,Diversity,pathogenicity,andmanagementofVerticilliumspecies.AnnualReviewofPhytopathology,2009,47:39-62.

13.Murashige,T.,Skoog,F.,Arevisedmediumforrapidgrowthandbioassayswithtobaccotissuecultures.Physiologiaplantarum,1962,15(3):473～497.

14.OrtinaCandCulianez-MaciaFA.,Tomatotransformationandtransgenicplantproduction.Plantcell,TissueandOrganCulture,2004,76:269-275.

15.PeggGF,BradyBL.,Verticilliumwilts.NewYork:CABIPublishing,2002,P432.

16.PowelsonML,RoweRC.,Biologyandmanagementofearlydyingofpotatoes.AnnualReviewofPhytopathology,1993,31:111-26.

17.RichardA.Jefferson,T.A.K.andBevan,M.W.,GUSfusions:B-glucuronidaseasasensitiveandversatilegenefusionmarkerinhigherplants.EuropeanMolecularBiologyOrganization,1987,6(13):3901-3907.

18.RoweRC,DavisJR,PowelsonML,RouseDI.,Potatoearlydying:causalagentsandmanagementstrategies.PlantDisease,1987,71:482-89.

19.RoweRC,PowelsonML.,Potatoearlydying:managementchallengesinachangingproductionenvironment.PlantDisease,2002,86:1184-93.

20.ShenkR.U.,HildebrandtA.C.,Mediumandtechniquesforinductionandgrowthofmonocotyledonousanddicotyledonousplantcellcultures.CanadianJournalofBotany,1972,50:199-204.

21.SubbaraoKV,HubbardJC,GreatheadAS,SpencerGA.,CompendiumofLettuceDiseases,1997,26-27.

22.TalboysPW.,Aculture-mediumaidingtheidentificationofVerticilliumalboatrumandV.dahlaie,PlantPathology,1961,5:57-58.

23.WeiguoFang,BoLeng,YuehuaXiao,KaiJin,JinchengMa,YanhuaFan,JingFeng,XingyongYang,YongjunZhang,andYanPei.,CloningofBeauveriabassianaChitinaseGeneBbchit1andItsApplicationToImproveFungalStrainVirulence.AppledandEnvironmentalMicrobiology,2005,363-370.

24.WeiguoFang,YongjunZhang,XingyongYang,XuelianZheng,HuiDuan,YiLi,YanPei.,

Agrobacteriumtumefaciens-mediatedtransformationofBeauveriabassianausinganherbicideresistancegeneasaselectionmarker.JournalofInvertebratePathology,2004,85:18–24.

25.Y.Bolek,A.A.Bell,K.M.El-Zik,P.M.ThaxtonandC.W.Magill.,ReactionofCottonCultivarsandanF2PopulationtoStemInoculationwithIsolatesVerticilliumdahliae.JournalofPhytopathology,2005,153:269-273.

26.ZhibingLuo,YongjunZhang,KaiJin,JinchengMa,XingWangandYanPei.,ConstructionofBeauveriaBassianaT-DNAinsertionmutantcollectionsandidentificationofthermosensitiveandosmosensitivemutants.ActaMicrobiologicaSinica,2009,49(10):1301-1305。

Claims

1. beauveria bassiana BbP4-ATPase gene is improving plant to the purposes in resistance to verticillium wilt, its

In by beauveria bassiana BbP4-ATPase gene integration target approach plant is built transgenic plant, and make described BbP4-ATPase gene express in plant and improve the resistance of plant to verticillium.

2. purposes as claimed in claim 1, the nucleotide sequence of wherein said beauveria bassiana BbP4-ATPase gene is as shown in SEQIDNO.15.

3. improve the method for plant to resistance to verticillium wilt, wherein by improving the resistance of described plant to verticillium at target plant expression in vivo foreign gene, described foreign gene is beauveria bassiana BbP4-ATPase gene.

4. method according to claim 3, comprises the steps:

5. method as claimed in claim 3, comprises the steps:

3) transgenic plant with the resisting verticillium of raising are obtained.

6. method according to claim 3, the nucleotide sequence of wherein said beauveria bassiana BbP4-ATPase gene is as shown in SEQIDNO.15.

7. the method as described in any one of claim 3 ~ 6, wherein said target plant is tomato, tobacco or cotton.

8. as claimed in claim 5 method, wherein step 1) described in recombinant plant expression vector there is structure as shown in Figure 8.

9. there is a preparation method for the transgenic plant of resistance to verticillium wilt, comprise the following steps:

10. preparation method as claimed in claim 9, the nucleotide sequence of wherein said beauveria bassiana BbP4-ATPase gene is as shown in SEQIDNO.15.